Materials for modulating biological responses and methods of making

ABSTRACT

A polymeric composition capable of releasing nitric oxide and modulating biological responses comprises a biocompatible polymer and S-nitrosated thiol bonded to the biocompatible polymer. The polymeric composition can have a thiol conversion of at least 40%. The polymeric composition can also have a nitric oxide recovery of at least 40% when under thermal decomposition conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/403,557, filed Feb. 23, 2012, entitled “MATERIALS FOR MODULATINGBIOLOGICAL RESPONSES AND METHODS OF MAKING” which claims priority toU.S. Provisional Application No. 61/446,121, filed Feb. 24, 2011,entitled “METHODS FOR MODULATING CELL RESPONSE,” the contents of whichare hereby incorporated by reference in their entirety.

GOVERNMENT LICENSE

The invention described herein was made with Government support underGrant No. W81XWH-11-2-0113 awarded by the Department of DefenseCongressionally Directed Medical Research Program (CDMRP). Thegovernment has certain rights in the invention.

TECHNICAL FIELD

The present invention relates generally to modulating biologicalresponses and materials for modulating such responses.

BACKGROUND

In the case of injury, whether a consequence of surgery or from anaccident or a mishap, wounds do not heal completely and this often leadsto additional health complications. Of the reasons for this is becausein would injuries, multiple biochemical pathways are activated and thusin order to achieve complete healing, simultaneous modulation ofmultiple biological responses is needed, in a similar way in manydisease sates, several different cell and protein types are affected. Totreat the disease effectively, all of the affected cell types must betreated. This involves developing methods and materials that caneffectively modulate multiple biological responses.

As one example, a recent study by the American Academy of OrthopedicSurgeons estimates that over 500,000 bone-grafting surgeries areperformed annually. By 2030, an overall incident increase of 600% ispredicted in the United States alone. Standard clinical graftingpractices to repair bone-tissue damage include autografting,allografting and xenografting. However, these procedures often result inincomplete healing and lead to additional health complications inpatients. To overcome these problems, researchers are developing newbone-tissue engineering strategies such as using natural and syntheticmaterials as scaffolds for repair. A number of these engineeredscaffolds are currently available for clinical uses. However, awell-accepted, versatile and clinically-proven scaffold is yet to befully realized.

Because of the multi-faceted problems associated with wound injuries, itwould be ideal for interventions to treat inflammation, thrombosis,infection, and wound healing in one treatment that can be easily appliedin a variety of field settings—emergency response, battlefield,hospitals, homes and clinics. An ideal treatment would inhibit multipleconsequences of injury, such as inflammation, thrombosis, and infection,without causing systemic effects. At the same time, it would bejudicious if the treatment also promoted wound healing processes. Inthese ways, a method is needed that can modulate multiple cellularresponses.

Such as strategy requires (1) the identification of suitable therapeuticagent(s) with short biological half-lives and (2) designer polymers thatact as sophisticated drug carriers. The therapeutic agents used todevelop these materials should be at least in part agents that arealready involved in normal homeostasis. For example, the endothelialcells that line all blood vessel walls have a number of boundtherapeutic agents and bioagents that are released from the surface ofthe endothelial cells that are responsible for maintaining normalhomeostasis within the blood. As a result, synthetic materials andmethods that have features that replicate the function of the normalendothelium are more likely to provide the ultimate route to safelymodulating biological responses. As such, the materials and methodsdescribed herein leverage the biological properties of naturallyoccurring biogents in synthetic materials to control biologicalresponses in order to treat a wide range of diseases or to preventbiofouling or treat injuries associated with a variety of medicaldevices where localized control of function is only at thefluid-delivery agent interface where action is targeted.

To date, two major classes of synthetic polymers have been explored asmaterials in these types of applications. The first class includessynthetic biodegradable polymers such as polylactide (PL), polyglycolide(PG), poly(lactide-co-glycolide) (PLGA) and poly(ε-caprolactone)(PC).These materials are formed into nanoscaffolds using the process ofelectrospinning. The resulting scaffolds have diameters between 50-500nm (similar to the size of many naturally occurring fibrous componentssuch as collagen within ECM), high porosities (up to 80%), and largesurface areas for cell attachment, bone in-growth, and nutrienttransport, making the materials a suitable ECM analogue for tissueengineering applications. However, because these polymers have relativelow hydrophilicities and lack cellular recognition, cell affinity to thescaffold to promote osteointegration is significantly diminished. In theend, the scaffold results in poor cell adhesion, migration,proliferation and differentiation.

A second class of biodegradable materials that have been studied forthese applications include polysaccharide-grafted polymers.Investigators have demonstrated that these materials have adequatemechanical properties (tensile strength and bending strength) to supporttissue growth over natural materials such as collagen. Moreover, thepolysaccharides materials have improved cell compatibility andstructural integration with many cell adhesion molecules and matrixglycoproteins as compared to the PL and PLGA polymers. For example,chitosan, a naturally available polysaccharide, is structurally similarto glycosaminoglycans (GAGs) present within bones and possesses a numberof osteophilic advantages including biocompatibility andbiodegradability. Similarly, dextran, a homopolymer of glucose withpredominantly α-(1→6) linkages has been investigated as a material fortissue scaffolds due to its relative biocompatibility and degradability.The high surface tension of these polysaccharide materials due to theirpolycationic nature have caused challenges in fabricating nanofibersusing electrospinning methods. As a result, composites materials ofchitosan have been prepared by blending the polysaccharide with variousfiber-forming polymers such as poly(vinyl alcohol), poly(ethyleneoxide), poly(vinyl pyrrolidone), poly(ε-caprolactone) andpoly(L-lactic-co-ε-caprolactone). The composite materials were thensuccessfully spun into nanofibers. The resulting nanofibers, however hadinconsistence mechanical and cell affinities in regions of the scaffold.

Although the synthetic and polysaccharide-derived materials havedemonstrated an alternative to autografting, allografting andxenografting, the materials still do not possess all of the requisitebiological properties of an ideal material to modulate cellularresponses. Specifically, the materials cause activation of thecoagulation cascade and provoke the immune response system (i.e., causeinflammation). As shown in FIG. 1, the tissue healing cascade beginsimmediately following injury and goes through four stages: hemostasis,inflammation, proliferation, and remodeling. First, the coagulationcascade is activated and platelets aggregate around exposed collagenleading to a fibrin clot matrix that leads to eventual healing.Subsequently, a variety of other factors are released includingcytokines, platelet-derived growth factor (PDGF) and transforming growthfactor-beta (TGF-β) during the inflammatory phase. As a result of thisrelease, neutrophils, macrophages, and lymphocytes are stimulated. Theproliferative phase follows and is marked by epithelialization,angiogenesis, and fibroblast growth and results in new connectivetissue. In the final phase collagen is cross-linked and scar maturationresults. If any part of the healing cascade is perturbed, fibrosis andchronic ulcers may result.

SUMMARY

In one embodiment, a polymeric composition capable of releasing nitricoxide and modulating biological responses comprises a biocompatiblepolymer and S-nitrosated thiol bonded to the biocompatible polymer. Thepolymeric composition can have a thiol conversion of at least 40%. Thepolymeric composition can also have a nitric oxide recovery of at least40% when under thermal decomposition conditions.

A method of modulating a biological response is also provided. Themethod includes contacting the subject with a medical device comprisinga polymeric material. The polymeric material includes a biocompatiblepolymer. Functional moieties are bound to the biocompatible polymer andare capable of releasing nitric oxide. The polymeric material has anitric oxide recovery of at least about 40% under thermal decompositionconditions.

A method of forming a biocompatible polymer capable of releasing nitricoxide for modulating biological responses is also provided. The methodincludes activating carboxyl groups of the biocompatible polymer in anon-aqueous solution by reaction with NHS and converting thiol residueson the biocompatible polymer to S-nitrosated residues under non-aqueousconditions, wherein the thiol conversion is at least about 40%

While multiple embodiments are disclosed, still other embodiments of thepresent invention will become apparent to those skilled in the art fromthe following detailed description, which shows and describesillustrative embodiments of the invention. Accordingly, the drawings anddetailed description are to be regarded as illustrative in nature andnot restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram of a tissue healing cascade.

FIG. 2 illustrates the synthesis of 5-nitrosated PLGH polymers.

FIG. 3 are SEM images of electrospun nanofibers of (A) PLGH, (B)PLGH-cysteamine, (C) PLGH-cysteine, (D) PLGH-homocysteine, (E)S-nitrosated PLGH-cysteamine, (F) S-nitrosated PLGH-cysteine, (G)S-nitrosated PLGH-homocysteine, (H) S-nitrosated PLGH-cysteamine afterNO release, 48 h, (I) S-nitrosated PLGH-cysteine after NO release, 48 h,and (J) S-nitrosated PLGH-homocysteine after NO release, 48 h.

FIG. 4 is a graph illustrating the real-time nitric oxide (NO release)profiles from individual S-nitrosated polymers using polymer films underphysiological conditions (10 mM PBS buffer/pH 7.4/37° C.) for 48 h.

FIG. 5 is a graph illustrating NO release profiles from individualelectrospun S-nitrosated polymers under physiological conditions (10 mMPBS buffer/pH 7.4/37° C.) for 48 h.

FIG. 6 illustrates a vitro degradation profile of PLGH polymers in 10 mMPBS buffer (pH 7.4, 37° C.).

FIG. 7 illustrates a synthesis of NO releasing diazeniumdiolatedPLGH-DETA.

FIG. 8 illustrates dextran modifications using reductive amination.

FIG. 9 illustrates dextran modifications using 4-nitrophenylchloroformate activation.

FIG. 10 illustrates a synthesis of a S-nitrosated dextran derivatives.

FIG. 11 is a graph illustrating a NO release profile of S-nitrosateddextran-cysteamine derivative (9a) in phosphate buffer saline (PBS, 10mM phosphate, pH 7.4) at 37° C.

FIG. 12 is a graph illustrating a NO release profile of S-nitrosateddextran-cysteine derivative (9b) in phosphate buffer saline (PBS, 10 mMphosphate, pH 7.4) at 37° C.

FIG. 13 illustrates a synthesis of a S-nitrosated dextran derivativesthrough carboxymethylation of dextran.

FIG. 14 is a graph illustrating a NO release profile of S-nitrosateddextran-cysteine (12b) under physiological conditions.

FIG. 15 illustrates a synthesis of a S-nitrosated chitosan derivative.

FIG. 16 is a graph illustrating a NO release profile from S-nitrosatedchitosan derivative in phosphate buffer saline (PBS, 10 mM phosphate, pH7.4) at 37° C.

FIG. 17 illustrates a synthesis of a PLGH-dextran conjugate.

DETAILED DESCRIPTION Definitions

For convenience, before further description of the present invention,certain terms used in the specification and examples are described here.These definitions should be read in light of the remainder of thedisclosure and understood as by a person of skill in the art. Also, theterms “including” (and variants thereof), “such as”, “e.g.”, “i.e.” asused herein are non-limiting and are for illustrative purposes only.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“Biodegradable” means chemical breakdown of materials by a physiologicalenvironment. This can include, but is not limited to physiologicalfluids such as blood and blood components, subcutaneous fluid, tissuefluid, or urine.

“Biological responses” refers to any biochemical pathways, cells,proteins, DNA, RNA, and other substances in the body that are altered.

“Host-guest” is a term that describes the relationship between adiscrete compound (“guest”) that is located within the pores or openspaces of a metal-organic compound (“host”). The discrete guest and themetal-organic compound in this relationship are not strongly covalentlybonded. In many cases, the discrete guest compound, such as carbondioxide gas, is stored in the pores and open spaces of the host, such asMOF compounds typically exhibit.

“Porosity” describes the size of the void spaces in a material. Thehigher the void space compared to material space, the higher theporosity. Porosity can range from 0-100%.

“Encapsulant” or “Encapsulating” refers to surrounding the agent inanother material.

“Secondary therapeutic agent” refers to compounds that cause a desirableand beneficial physiological result in response to the compound.Exemplary non-genetic therapeutic agents for use in conjunction with thepresent invention include, but are not limited to: (a) anti-thromboticagents such as heparin, heparin derivatives, urokinase, and PPack(dextrophenylalanine proline arginine chloromethylketone); (b)anti-inflammatory agents such as dexamethasone, prednisolone,corticosterone, budesonide, estrogen, sulfasalazine and mesalamine; (c)antineoplastic/antiproliferative/anti-miotic agents such as paclitaxel,5-fluorouracil, cisplatin, vinblastine, viricristine, epothilones,endostatin, angiostatin, angiopeptin, monoclonal antibodies capable ofblocking smooth muscle cell proliferation, and thymidine kinaseinhibitors; (d) anesthetic agents such as lidocaine, bupivacaine andropivacaine; (e) anti-coagulants such as D-Phe-Pro-Arg chloromethylketone, an RGD peptide-containing compound, heparin, hirudin,antithrombin compounds, platelet receptor antagonists, anti-thrombinantibodies, anti-platelet receptor antibodies, aspirin, prostaglandininhibitors, platelet inhibitors and tick antiplatelet peptides; (f)vascular cell growth promoters such as growth factors, transcriptionalactivators, and translational promoters; (g) vascular cell growthinhibitors such as growth factor inhibitors, growth factor receptorantagonists, transcriptional repressors, translational repressors,replication inhibitors, inhibitory antibodies, antibodies directedagainst growth factors, bifunctional molecules consisting of a growthfactor and a cytotoxin, bifunctional molecules consisting of an antibodyand a cytotoxin; (h) protein kinase and tyrosine kinase inhibitors(e.g., tyrphostins, genistein, quinoxalines); (i) prostacyclin analogs;(j) cholesterol-lowering agents; (k) angiopoietins; (l) antimicrobialagents such as triclosan, cephalosporins, aminoglycosides andnitrofurantoin; (m) cytotoxic agents, cytostatic agents and cellproliferation affectors; (n) vasodilating agents; (o) agents thatinterfere with endogenous vasoactive mechanisms; (p) inhibitors ofleukocyte recruitment, such as monoclonal antibodies; (q) cytokines; (r)hormones; (s) inhibitors of HSP 90 protein (i.e., Heat Shock Protein,which is a molecular chaperone or housekeeping protein and is needed forthe stability and function of other client proteins/signal transductionproteins responsible for growth and survival of cells) includinggeldanamycin, (t) alpha receptor antagonist (such as doxazosin,Tamsulosin) and beta receptor agonists (such as dobutamine, salmeterol),beta receptor antagonist (such as atenolol, metaprolol, butoxamine),angiotensin-II receptor antagonists (such as losartan, valsartan,irbesartan, candesartan and telmisartan), and antispasmodic drugs (suchas oxybutynin chloride, flavoxate, tolterodine, hyoscyamine sulfate,diclomine), (u) bARKct inhibitors, (v) phospholamban inhibitors, (w)Serca 2 gene/protein, (x) immune response modifiers includingaminoquizolines, for instance, imidazoquinolines such as resiquimod andimiquimod, and (y) human apolioproteins (e.g., AI, AII, AIII, AIV, AV,etc.). Exemplary genetic therapeutic agents for use in conjunction withthe present invention include anti-sense DNA and RNA as well as DNAcoding for the various proteins (as well as the proteins themselves):(a) anti-sense RNA, (b) tRNA or rRNA to replace defective or deficientendogenous molecules, (c) angiogenic and other factors including growthfactors such as acidic and basic fibroblast growth factors, vascularendothelial growth factor, endothelial mitogenic growth factors,epidermal growth factor, transforming growth factor ÿ and ÿ,platelet-derived endothelial growth factor, platelet-derived growthfactor, tumor necrosis factor a, hepatocyte growth factor andinsulin-like growth factor, (d) cell cycle inhibitors including CDinhibitors, and (e) thymidine kinase (“TKE”) and other agents useful forinterfering with cell proliferation. Also of interest is DNA encodingfor the family of bone morphogenic proteins (“BMP's”), including BMP-2,BMP-3, BMP-4, BMP-5, BMP-6 (Vgr-1), BMP-7 (OP-1), BMP-8, BMP-9, BMP-10,BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, and BMP-16. Currently preferredBMP's are any of BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7. Thesedimeric proteins can be provided as homodimers, heterodimers, orcombinations thereof, alone or together with other molecules.Alternatively, or in addition, molecules capable of inducing an upstreamor downstream effect of a BMP can be provided. Such molecules includeany of the “hedgehog” proteins, or the DNA's encoding them.

“Material” and “matrix” are art recognized and refer to that themetal-organic compounds, therapeutic agents, and secondary therapeuticagents that are contained within. These can include, but are not limitedto, plastics, cements, and clays.

“Polymer” is a large molecule composed of repeating structural orconstitutional units, usually referred to as monomers, connected bycovalent chemical bonds. Polymers can consist of the same or differingrepeat units in order or random fashion. Polymers may take on a numberof configurations, which may be selected, for example, from cyclic,linear, branched and networked (e.g., crosslinked) configurations.Branched configurations include star-shaped configurations (e.g.,configurations in which three or more chains emanate from a singlebranch point, such as a seed molecule), comb configurations (e.g.,configurations having a main chain and a plurality of side chains),dendritic configurations (e.g., arborescent and hyperbranched polymers),and so forth. As used herein, “homopolymers” are polymers that containmultiple copies of a single constitutional unit. “Copolymers” arepolymers that contain multiple copies of at least two dissimilarconstitutional units, examples of which include random, statistical,gradient, periodic (e.g., alternating), and block copolymers.

“Plasticizer” is art recognized is a compound that is added to polymersthat increases the plasticity or fluidity of the material to which theyare added. Some examples include, but are not limited to,dicarboxylic/tricarboxylic ester-based plasticizers such as dioctylsebacate (DOS), benzoates, sulfonamides, organophosphates, and glycols,or benzoates.

The terms “blend”, “layered”, “hybrid”, “composite”, and“hybrid-composite” are used to describe materials that are made frommore than one component and in various combinations.

“Interpenetrating network” (IPN) describes any material containing twomacromolecular compounds, such as polymers or metal-organic compoundswhose structures interweave and fill the spaces of the other substanceby physical means. These networks may or may not have chemicalinteractions with each other.

“Crystallinity” is art recognized and refers to the ordered structure ofthe polymer.

“Producing” is a generic term used to describe all mechanisms ofdelivery including generating and releasing modes.

“Biocompatibility” is a generic term that describes an interaction orrelationship with physiological or biological systems.

“Medical device” refers to product which is used for medical purposes inpatients, in diagnosis, therapy, treatment, or surgery. If applied tothe body, the effect of the medical device can be physical or chemical.

“Bioerodable” refers to the chemical breakdown of material by thephysiological environment beginning at the surface of the material.

“Physiological fluid” refers to any fluid produced by the body,including but not limited to, subcutaneous fluid, saliva, blood,extracellular fluid, and urine.

“Processing” refers to manipulation of the material by a physical means.This includes but is not limited to spray coating, extrusion, dipcoating, molding, electrospinning, and casting.

“Thermal decomposition conditions” refers to raising the temperature ofa sample until all of the nitric oxide releasing moieties havedecomposed as evidenced by a baseline NO measurement.

Biodegradable Polymers Capable of Modulating Biological Responses

The present invention includes biodegradable polymers comprised offunctional moieties bound to the polymeric backbone that are capable ofmodulating biological responses, for example, but not limited to, byproducing NO. Other examples include the use of other naturallyoccurring biological agents such as PGI₂, thrombomodulin and the like.These functional biological agents can be included into thebiodegradable materials as small organic compounds, metal organicframeworks, inorganic compounds, or by non-covalent attachment means

Additional embodiments include the combination of polymeric compoundswith and without secondary therapeutic agents that are capable ofmodulating biological responses as well.

Still further embodiments describe compositions of materials comprisedof polylactide/polygycolide co-polymers and polysaccharides boundpolymers combined with functional groups capable of releasing NO and/orother naturally occurring biological agents such as PGI₂, thrombomodulinand the like under physiological conditions that modulate biologicalresponses

Cells are disrupted when an injury occurs. Previously, drugs ortherapeutic agents were provided to injuries in order to preventundesired biological processes. The current invention uses naturallyoccurring therapeutic agents or biological agents, such as nitric oxide,in concert with appropriate delivery platforms to provide an effectivesolution to modulating multiple biological responses, such as cellularand protein behavior, in the treatment of wounds. The current inventionprovides a therapeutic agent to help damaged cells function properlyuntil they grow back to their healthy function. These goals are achievedby providing a material having a high therapeutic loading and having ahigh therapeutic agent recovery percentage.

Suitable delivery platforms include biodegradable polymers such aspolylactide (PL), polyglycolide (PG), poly(lactide-co-glycolide) (PLGA)and poly(ε-caprolactone)(PC). These materials are made by incorporatingmonomeric units in appropriate ratios that give rise to morphologicaland biochemical properties. These monomeric units can be postsynthetically modified to contain additional therapeutic action. As anexample, diazeniumdiolates and S-nitrosothiols are formed on nitrogenand sulfur-based functional groups. The monomer units used to create thebiodegradable polymer can be readily modified to incorporate thenecessary nitrogen and sulfur based linkers required to allow thestructures to be generated that can store and release NO inphysiological systems. If multiple therapeutic agents are used, thetherapeutic agents can be in the same material layer or differentmaterial layers. Not all of the material layers used need to have atherapeutic agent. Each layer of the overall material may contain aunique combination of zero, one, or multiple therapeutic components.Other suitable polymers include polysaccharides, such as but not limitedto dextran and chitosan.

Nitric oxide (NO) is a well known naturally-occurring biological agentresponsible for maintaining normal hemostasis, cellular signaling, andbone development in the body as well as promoting healthy cell growthand wound healing. Nitric oxide is also responsible for preventingplatelet activation and adhesion as well as microbial growth andbacterial invasion of tissue. Nitric oxide also serves as an effecter inwound healing mechanisms, and regulates angiogenesis andrevascularization. Most recently, published reports have demonstratedthat NO produced by the endothelial NO synthase (eNOS) also plays animportant role in bone development and healing as an inducer ofosteoblastic differentiation (bone formation) especially in mesenchymalprogenitor cells. Mesenchymal progenitor cells promote mineralization,stimulate expression in fibroblasts, enhancing osteoclast activities andinhibiting bone resorption. Moreover, alleviation of osteoporotic boneloss by administration of exogenous NO donors provided a better choicefor osteoporosis therapy compared to conventional therapies. Previouswork has shown that functional moieties capable of releasing NO in vivoincrease the hemocompatibility of materials by reducing thethrombogenicity. In addition, the release of NO reduces the inflammatoryresponse towards the implanted materials. Further, nitric oxide hasmultiple biphasic effects on cells. For example, nitric oxide's role intumor biology includes angiogenesis and metastasis, and modulation ofboth necrosis and apoptosis. Thus, nitric oxide has the capability oftreating abnormal cell growth while at the same time promoting thehealing of surrounding cells. Due to nitric oxide's multiple effects ofcells, the incorporation of functional moieties capable for releasingnitric oxide under physiological conditions can impart severaladvantages to modulating multiple biological processes resulting inhealthy incorporation of medical devices into the body, reduction ininfection, thrombosis, and monocyte activation, improvement in woundhealing at the site of injury, and metathesis of cancerous cells.

As discussed above, the polymeric composition is capable of releasingnitric oxide and modulating biological responses. The polymericcomposition may be capable of modulating biological responses at leastin part due to the polymeric composition having a high nitric oxideloading. For example, the polymeric composition may have a nitric oxideloading greater than 0.1 mmol/g polymer, greater than 0.2 mmol/gpolymer, greater than 0.3 mmol/g polymer, greater than 0.4 mmol/gpolymer or greater than 0.5 mmol/g polymer. The high nitric oxideloading enables nitric oxide release over a longer time frame. In oneexample, nitric oxide is released over a period of at least 172 hours.The nitric oxide loading and release time frame enable therapeuticdosages of nitric oxide that modulate biological responses, not justinhibit consequences of injury (e.g., inflammation, thrombosis, andinfection).

The polymeric composition also has a relatively high nitric oxiderecovery percentage, which can be calculated by measuring the nitricoxide released during a given period compared to the equivalent nitricoxide present in the initial polymer. Suitable polymeric compositionshave a nitric oxide recovery of at least about 40%, at least about 50%,at least about 60%, at least about 70% or at least about 80%, underthermal dry conditions (step-wise heating up to 100° C. until base linelevels of NO are reached). Under physiological conditions (10 mM PBSbuffer, pH 7.4, 37° C.) for 48 hours, suitable polymeric compositionshave a nitric oxide recovery of at least about 20%, at least about 25%,at least about 30%, at least about 40% or at least about 50%. Underphysiological conditions (10 mM PBS buffer, pH 7.4, 37° C.) suitablepolymeric compositions have a nitric oxide recovery of at least 70%, atleast 75%, or at least 80% if allowed to go to competition.

In one example the polymeric composition includes a biocompatiblepolymer, such as a biodegradable polymer or a polysaccharide, andS-nitrosated thiol bonded to the biocompatible polymer. The polymericcomposition can be synthesized to include thiol residues. In one exampleat least 40% of the thiol residues are converted to S-nitrosated thiol(e.g., a thiol conversion of at least 40%). In another example, thepolymeric compositions has a thiol conversion of at least 50%, 60%, 70%,80%, 90%. In other examples the thiol conversion can be as great as100%.

In one example, the thiol residue is selected from the group consistingof residue of a thiol or an amino-thiol. In another example the thiolresidue is selected from the group consisting of cysteamine, cysteine,and homocysteine residues and combinations thereof. In a still furtherexample, the thiol residue is selected from the group consisting ofglutathione or penicillamine.

Methods of making suitable polymeric compositions are also provided. Inone example, the polymeric compositions are synthesized with anon-aqueous method. For example, the synthesis method may includeactivating carboxyl groups of the biocompatible polymer in a non-aqueoussolution by reaction with N-hydroxysuccinimide (NHS), adding a thiol tothe biocompatible polymer to attach thiol residues onto the backbone ofthe biocompatible polymer, and converting the thiol residues toS-nitrosated residues under non-aqueous conditions. In one example atleast about 40% of the thiol residues are converted to S-nitrosatedresidues (i.e., a thiol conversion of at least about 40%).

After synthesis, the polymeric compositions can be processed into afinal form, such as a polymer film, nanofibers, or multilayer coatings.Typically, polymeric compositions can lose nitric oxide capacity duringprocessing. The polymers and their processing in these ways remainstable. Processing the polymeric compositions into different engineeredforms, achieves a higher NO release rate than previously believedpossible. The time period over which the NO is released from theprocessed polymeric compositions is also longer than previouslyachieved. This may be due to higher surfaces areas, changes in surfacemorphology or wettability, access of fluids (either water or otherphysiological fluids (i.e., blood, stomach fluid, interstitial fluid,etc.) or cells to the material interface.

In one example, polymeric materials prepared above can be electrospuninto fibers. In brief, polymer solutions in a suitable solvent areejected from the tip of a fine nozzle of a syringe, maintained at a highDC potential. Using a syringe pump, a constant flow of the fluid will bemaintained and once the electrostatic repelling force of the chargesovercomes the surface tension of the solution droplet, the charges leavethe droplet and drag the polymer into thin fibers. Finally because ofunstable whip-like motions, these fibers will further reduce theirdiameter and elongate into nanofibers.

The polymeric materials can be electrospun to produce micro andnanoscaffolds for treating bone repair injuries or tissue injuries withenhanced bone development and healing capability with distinct andmultifaceted mode of actions. Scanning electron micrographs (SEMs)demonstrating the ability to electrospin the materials are given in FIG.3.

The polymeric materials can be prepared as biodegradable transdermalpatches for wound healing.

The polymeric materials can be prepared as biodegradableanti-inflammatory surgical threads or other implanted devices such asstents.

The polymeric materials can be processed into macrocapsules ormicrospheres. One such strategy is to dissolve S-nitrosated dextranderivatives in a suitable solvent like dimethyl sulfoxide (DMSO) andthen emulsified in a large excess of non-soluble solvent such as hexane,heptane etc. in presence of a suitable emulsifying agent such astween-20.

Further, in a composition embodiment, the therapeutic biodegradablematerials are incorporated into another material, such as a polymer. Insome embodiments, the material can be a polymer that has additionalchemical functionalities such as, but not limited to, amine, carboxyl,halide, ketone, urethane, urea, silicone, or aldehyde groups. Thematerial can also have differing degrees of porosity or diffusioncharacteristics. Other compositions of the invention includeencapsulating the biodegradable polymers into other materials such aspolymers or using the biodegradable polymer as encapsulants for othertherapeutics (i.e., drugs). These drugs may have the form of a metalorganic framework or an organic structure. Other compositions mayinclude adding a secondary therapeutic agent to the material in concertwith the biodegradable polymer that produces nitric oxide. The secondarytherapeutic agent could be covalently attached to the metal-organiccompound, blended with the biodegradable polymer, or blended intoanother material such as an organic polymer. The secondary therapeuticagent may also be covalently attached to the biodegradable polymer ornon-specifically bound to the biodegradable polymer.

Additional compositions may include, but are not limited to, using thebiodegradable polymers in conjunction with synthetic polymers such aspolyurethane (PU), polyesters, polyethers, silicones, silicates,poly(vinyl chloride) (PVC), acrylates, methacylates, dextran, syntheticrubber (cis-polyisoprene), polyvinyl acetate, Bakelite, polychloroprene(neoprene), nylon, polystyrene, polyethylene, polypropylene,polyacrylonitrile, polyvinyl butyral (PVB), poly(vinylidene chloride),or fluorinated polymers such as polytetrafluoroethylene (PTFE) whereeither or both of the metal-organic compounds and secondary therapeuticagents are incorporated. These synthetic materials can be used ashomopolymers or multi-component polymers (i.e., copolymer, tri-polymers,etc.). The polymers may be hydrophobic or hydrophilic or contain regionsof both hydrophobicity and hydrophilicity.

The composition can also include natural polymers such as DNA,phosphodiesters, polysaccharides, or glycosides where either or both ofthe therapeutic biodegradable polymers and secondary therapeutic agentsare incorporated.

The composition may also include other biodegradable or bioerodablepolymers in whole or in part of the final material formulation. Exampleof biodegradable or bioerodable polymers may include, but are notlimited to, polyesteramides, polyglycolide, polyanhydrides,polyorthoesters, ureas, urethanes, esters, ethers, polyhydroxybutyrate(PHB), polyhydroxyvaleratepolylactide, poly(-caprolactone),polyiminocarbonate, poly(dioxanone), polyarylates, as well as copolymersof these and other monomers such as -caprolactone with dl-lactide,-caprolactone with glycolide, lactide with glycolide, and glycolide withtrimethylene carbonate (TMC) Amino acid based polymers such astyrosine-derived polycarbonates are also included.

The composition may also include fibrous matrices, composite materials,layering, particles or blends. Nano- or micro-particles of the matrixmaterial incorporated with secondary therapeutic agents used eitheralone or in combination with another matrix material are included. Forexample, the therapeutic biodegradable polymer can be encapsulated intoa biodegradable cellulose material which is then delivered orally or isblended into another polymer matrix such as polyurethane and used as apermanent coating implant.

The polymeric materials can be further blended with plasticizers to makecomposite compositions to tailor the release rates and elasticity of thefinal polymeric material.

Compositions, configurations, and uses of this invention have themechanical properties that match the application such that the materialin its final embodiment remains sufficiently strong until thesurrounding tissue has healed, does not invoke an inflammatory or toxicresponse, and for biodegradable application is metabolized in the bodyafter fulfilling its purpose, leaving no trace. Further, the material iseasily processed into the final product form, demonstrates acceptableshelf life, and is sterilized by acceptable methods such as ethyleneoxide, gamma, or auto-clave.

Matrix materials for use in this invention can have various degrees ofcrystalline or amorphous character, ranging from 100% crystalline to100% amorphous. The material can have a range of differing microdomainsand morphologies that aid in the final application. The matrix materialscan also be used in this invention regardless of their stereochemistry.Both region- and stereoisomerization forms of the matrix material can beused. For example, the invention can be practiced using polymers withmultiple stereochemical forms such as isotactic, syndiotactic, andatactic.

Also included in this disclosure is a method for treating diseases,disorders, or conditions in a patient using therapeutically effectiveamounts of the described composition. These diseases, disorders, orconditions may be present in the patient prior to treatment as well ascaused by the procedure or the placement of the medical device. As usedherein, “treatment” refers to the prevention of a disease or condition,the reduction or elimination of symptoms associated with a disease orcondition, or the substantial or complete elimination of a disease orcondition.

Diseases, disorders or conditions may include, but are not limited to,tumors, organs including the heart, coronary and peripheral vascularsystem (referred to overall as “the vasculature”), the urogenitalsystem, including kidneys, bladder, urethra, ureters, prostate, vagina,uterus and ovaries, eyes, ears, spine, nervous system, lungs, trachea,esophagus, intestines, stomach, brain, liver and pancreas, skeletalmuscle, smooth muscle, breast, dermal tissue, cartilage, tooth, bone,local infection, systemic infection, biofouling, macrophage formation,heart disease, artery and vein damage or disease, tissue injury,vascular legions, initimal hyperplasia, heart failure, high or elevatedblood pressure, vasoconstriction, platelet adhesion, plateletaggregation, atherosclerosis, thrombo-embolism, thrombosis, smoothmuscle cell proliferation, sepsis, complications with medical devices,wounds caused by initicions or insertion of medical devices, cicatrices,endothelial cell damage, arrhythmias, heart defects, congenital heartdefects, cell overgrowth, and soft bones. The compositions describedherein can be used to promote angiogenesis, delivery of analytes to thesite of injury, and perfusion of blood to the site of injury. Thecompositions can also be used to regulate the coagulation cascade.

Compositions further include, but are not limited to, administration asclinically prescribed including intravenously, orally, bucally,parenterally, inhalation spray, topically either within conjunction witha delivery vehicle (i.e., bandage or gel) or alone, locally, andtransdermally.

Local delivery includes any means by which the composition can be madein contact with the targeted delivery site in the patient including, butnot limited to, sutures, bandages, patches, wraps, vascular implants,stents, drug pumps, catheters, guidewires or any other implantablemedical devices.

The disclosure also includes a medical device that comprises thecomposition described herein. Such a medical device includes, but arenot limited to, catheters (e.g., urological or vascular catheters suchas balloon catheters and various central venous catheters), guide wires,balloons, filters (e.g., vena cava filters and mesh filters for distilprotection devices), stents (including coronary vascular stents,peripheral vascular stents, cerebral, urethral, ureteral, biliary,tracheal, gastrointestinal and esophageal stents), stent coverings,stent grafts, vascular grafts, abdominal aortic aneurysm (AAA) devices(e.g., AAA stents, AAA grafts), vascular access ports, dialysis ports,embolization devices including cerebral aneurysm filler coils (includingGuglilmi detachable coils and metal coils), embolic agents, hermeticsealants, septal defect closure devices, myocardial plugs, patches,pacemakers, lead coatings including coatings for pacemaker leads,defibrillation leads, and coils, ventricular assist devices includingleft ventricular assist hearts and pumps, total artificial hearts,shunts, an interventional cardiology device, valves including heartvalves and vascular valves, anastomosis clips and rings, cochlearimplants, tissue bulking devices, and tissue engineering scaffolds forcartilage, bone, skin and other in vivo tissue regeneration, sutures,suture anchors, tissue staples and ligating clips at surgical sites,cannulae, metal wire ligatures, urethral slings, hernia “meshes”,artificial ligaments, orthopedic prosthesis such as bone grafts, boneplates, joint prostheses, orthopedic fixation devices such asinterference screws in the ankle, knee, and hand areas, tacks forligament attachment and meniscal repair, rods and pins for fracturefixation, screws and plates for craniomaxillofacial repair, dentalimplants, plastic tubing, a dialysis bag or membrane, a ventricularshunt, an external device applied directed to the skin as well asvarious other devices that are implanted or inserted into the body andfrom which therapeutic agent is released.

The medical device made be coated or completely fabricated from thematrices.

As discussed above, the composition may be incorporated into atissue-engineered scaffold. The tissue-engineered scaffold can providemechanical support for tissue growth coupled with naturally producedbiological mediators that promote healing. As such, the scaffold can beporous for nutrient transport, hydrophilic for cell attachment,biodegradable as the native tissue integrates, encourage bone cellmigration into a defect (osteoconduction), support and promoteosteogenic differentiation (osteoinduction), enhance cellular activitytowards scaffold-host tissue integration (osteointegration), present aphysicochemical biomimetic environment and actively promote or preventdesirable and undesirable physiological responses. Further the scaffoldmaterial can mimic the nanotopography and functionality of theextracellular matrix (ECM) and make use of biologically derivedmediators to successfully regenerate damaged tissue.

EXAMPLES

The present invention is more particularly described in the followingexamples that are intended as illustrations only, since numerousmodifications and variations within the scope of the present inventionwill be apparent to those of skill in the art. Unless otherwise noted,all parts, percentages, and ratios reported in the following examplesare on a weight bases, and all reagents used in the examples wereobtained, or are available, from the chemical suppliers described below,or may be synthesized by conventional techniques.

Example 1 Nitric oxide releasing S-nitrosatedpoly(lactic-co-glycolic-co-hydroxymethyl propionic acid) (PLGH) polymers

FIG. 2 is a scheme for preparing S-nitrosated PLGH polymers.

PLGH (1): L-lactide (1.7 g, 85% w/w), glycolide (0.2 g, 10% w/w),2,2-bis(hydroxymethyl)-propionic acid (HMPA, 0.1 g, 5% w/w) and stannousoctoate (0.01 g, 5% w/w of total polymer) were mixed with 2 mL drydichloromethane (DCM, freshly distilled using calcium hydride) in apolymerization tube under nitrogen. Solvent was removed under vacuum andapplied nitrogen and vacuum alternatively several times to remove anyadhered moisture before heating the mass to 70° C. in an oil bath.Vacuum was again applied for half hour at 70° C. and then replaced withnitrogen and heated to 120° C. to perform the polymerization in the meltphase. After 24 hour, the polymerized material was cooled to roomtemperature and crystallized using dichloromethane-diethyl ether mixture(1:20) to remove any unreacted monomers. Yield 1.67 g (83.5%). ¹H NMR(CDCl₃): δ 5.17 (m, —O—CH(CH₃)—CO—), 1.56 (d, —O—CH(CH₃)—CO—), 4.58-4.92(m, —O—CH—CO—), 4.18-4.40 (m, —O—CH₂—C—) and 1.27 (s, —CH₂—C(CH₃)—).

PLGH-cysteamine (2a): Carboxyl functionalized PLGH intermediate (1) (1.0g, 0.62 mmol COOH g⁻¹ polymer) and N-hydroxysuccinimide (NHS, 0.18 g,1.55 mmol, 2.5 molar eq.) were mixed together in 4 mL anhydrousdimethylformamide (DMF) under an N₂ atmosphere. A solution of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl,0.3 g, 1.55 mmol, 2.5 molar eq.) in DMF (6 mL) was slowly added using apressure-equalized addition funnel and maintained the mass at 20° C. for24 hour to complete the activation of all available carboxyl groups.0.35 g cysteamine hydrochloride (3.1 mmol, 5 molar eq. of COOH) wasdried separately in 12 mL anhydrous DMF under vacuum for two hour toremove any moisture and then mixed with triethylamine (0.47 g, 4.68mmol, 1.6 molar eq. of cysteamine hydrochloride). The resultedneutralized cysteamine was slowly charged into the NHS activated polymerunder N₂ and further stirred for 48 hour. The polymer solution was thenconcentrated under vacuum to remove DMF and the crude product wasprecipitated out by adding excess diethyl ether and dried under vacuum.The crude product was then redissolved in DCM (25 mL) and washed twotimes with saturated sodium chloride solution (5 mL) to remove excesscysteamine and to hydrolyze unreacted NHS ester groups. The clear DCMextract was separated and stirred with 50 mg dithiothreitol (DTT) forhalf hour to reduce any possibly formed disulfide bonds. The DCM extractwas again washed two times with saturated sodium chloride solution (5mL), demoisturised using anhydrous sodium sulphate and filtered throughcelite to remove any undissolved particles. The product was isolatedafter distilling the solvent and crystallized using excess diethylether. Yield 0.96 g (60%). ¹H NMR (CDCl₃): δ 3.09 (m, —NH—CH₂—CH₂—) and2.64 (m, —NH—CH₂—CH₂—).

PLGH-cysteine (2b): Experiments were performed following the methodgiven for 2a using a mixture of 0.38 g vacuum dried cysteine (3.1 mmol,5 molar eq.) and 0.16 g triethylamine (1.55 mmol, 2.5 molar eq.) inanhydrous DMF. Yield 0.6 g (60% w/w). ¹H NMR (CDCl₃): δ 5.14 (m,—OCH(CH₃)—CO—), 1.55 (d, —O—CH(CH₃)—CO—), 4.57-4.89 (m, —O—CH—CO—),4.23-4.39 (m, —O—CH₂—C—), 1.27 (s, —CH₂—C(CH₃)—) and 3.07 (m,—NH—CH₂—SH).

PLGH-homocysteine (2c): Experiments were performed following the methodgiven for 2a and 2b using a mixture of 0.44 g vacuum dried homocysteine(3.1 mmol, 5 molar eq.) and 0.16 g triethylamine in anhydrous DMF. Yield0.6 g (60% w/w). ¹H NMR (CDCl₃): δ 5.17 (m, —O—CH(CH₃)—CO—), 1.56 (d,—O—CH(CH₃)—CO—), 4.58-4.92 (m, —O—CH—CO—), 4.18-4.40 (m, —O—CH₂—C—),1.27 (s, —CH₂—C(CH₃)—), 2.12 (m, —NH—CH₂—CH₂—) and 2.51 (m,—NH—CH₂—CH₂—).

S-nitrosated PLGH-cysteamine (3a): 50 mg PLGH-cysteamine (2a) wasdissolved in 2 mL dichloromethane-methanol (1:2) mixture in an ambercolored EPA vial (Fisher Scientific, NJ). In a separate vial, a solutionof 8.4 mg t-butyl nitrite (4 molar eq., pre-treated with 10% w/vdisodium ethylenediamine tetraacetate dehydrate (EDTA—disodium salt)) in1 mL dichloromethane-methanol (1:2) mixture was prepared and added intothe polymer solution protected from direct exposure to light. Thesolution was stirred at 20° C. for 4 hour and then concentrated undervacuum to isolate the S-nitrosated product. UV-vis λmax (2 MeOH: 1 DCM):338 nm (ε=766.0±19.7 M⁻¹ cm⁻¹).

S-nitrosated PLGH-cysteine (3b): A 50 mg sample of PLGH-cysteine (2b)was dissolved in a 2 mL methanol:dichloromethane (2:1) mixture in anamber colored EPA vial. In a separate vial, a solution of 8.4 mg t-butylnitrite (4 molar eq., pretreated with 10% w/v EDTA disodium salt) in 1mL methanol:dichloromethane (2:1) mixture was prepared and added intothe polymer solution while protected from direct exposure to light. Thesolution was stirred at 20° C. for 8 h and then concentrated undervacuum to isolate the S-nitrosated product. UV-vis λmax (2 MeOH: 1 DCM):335 nm (8=882.9±18.2 M⁻¹ cm⁻¹).

S-nitrosated PLGH-homocysteine (3c): A 50 mg sample of PLGH-homocysteine(2c) was dissolved in a 2 mL methanol:dichloromethane (2:1) mixture inan amber colored EPA vial. In a separate vial, a solution of 8.4 mgt-butyl nitrite (4 molar eq., pre-treated with 10% w/v EDTA disodiumsalt) in 1 mL methanol:dichloromethane (2:1) mixture was prepared andadded into the polymer solution while protected from direct exposure tolight. The solution was stirred at 20° C. for 4 h and then concentratedunder vacuum to isolate the S-nitrosated product. UV-vis λmax (2 MeOH: 1DCM): 336 nm (ε=652.1±16.7 M⁻¹ cm⁻¹).

Example 2 Processed S-Nitrosated PLGH Polymers

The S-nitrosated PLGH polymers of Example 1 were processed into apolymer film or a nanofiber.

S-nitrosated polymer films were prepared on pre-cleaned glass slides byspin-coating a 100 mg/mL polymer solution in dry dichloromethane at aspeed of 5000 rpm for one minute using SCS spin coater (Spincoat G3P-8),shielded from direct exposure to light.

Nanofibers were prepared as follows. PLGH, its thiolated derivatives,and the S-nitrosated derivatives were dissolved in a 75:25 w/w mixtureof THF and DMF to obtain 10-40 w/w % solutions. The polymer solution wasdrawn into nanofibers by using electrospinning process. A 1 mL syringewith a 22 G blunt tip needle was loaded with polymer solution andinserted into a variable speed syringe pump (Kent Scientific Corp.,Torrington, Conn., USA) with a flow rate of 0.2 mL h⁻¹. A 10 kVpotential was applied via a high-voltage power supply (Gamma HighVoltage Research, Ormond Beach, Fla., USA). Fibers were collected onglass slides attached to a grounded copper plate. Fiber morphology wasdetermined by scanning electron microscopy (JEOL JSM-6500F, JEOL USA,Peabody, Mass., USA), after sputter coating with 10 nm of gold prior toanalysis.

FIG. 3 is SEM images of electrospun nanofibers of (A) PLGH, (B)PLGH-cysteamine, (C) PLGH-cysteine, (D) PLGH-homocysteine, (E)S-nitrosated PLGH-cysteamine, (F) S-nitrosated PLGH-cysteine, (G)S-nitrosated PLGH-homocysteine, (H) S-nitrosated PLGH-cysteamine afterNO release, 48 h, (I) S-nitrosated PLGH-cysteine after NO release, 48 h,and (J) S-nitrosated PLGH-homocysteine after NO release, 48 h.

Example 3 NO Release from the Processed S-Nitrosated PLGH Polymers

Real-time NO release from the processed S-nitrosated PLGH polymers ofExample 3 was analyzed using Sievers chemiluminescence NO Analyzers®(NOA 280i, GE Analytical, Boulder, Colo., USA). The instrument wascalibrated before each analysis using nitrogen as the zero gas and a 45ppm NO gas. NO release from the S-nitrosated polymer samples wasanalyzed in deoxygenated 10 mM PBS buffer (pH 7.4) at 37° C., shieldedfrom direct exposure to light. NO release measurements were recordedfrom all S-nitrosated PLGH polymers using both polymer film andnanofiber forms. Experiments were repeated in triplicate at a datainterval of 5 sec at a sampling rate of 200 mL min⁻¹ with a cellpressure of 9.7 Torr and an oxygen pressure of 6 psig. Representative NOrelease profile from the polymer film and nanofiber forms in PBS (10 mM,pH 7.4) and 37 degrees Celsius are given in FIGS. 4 and 5 respectively.

In FIG. 4, line 110 represents the NO release profile from thenitrosated PLGH-cysteine polymer in film form, line 112 represents theNO release profile from the nitrosated PLGH-cysteamine polymer in filmform, and line 114 represents the NO release profile from the nitrosatedPLGH-homocysteine polymer in film form.

In FIG. 5, line 116 represents the NO release profile from electrospunnitrosated PLGH-cysteine polymer, line 118 represents the NO releaseprofile from electrospun nitrosated PLGH-cysteamine polymer, and line120 represents the NO release profile from electrospun nitrosatedPLGH-homocysteine polymer. All polymers in FIGS. 3 and 4 were testedunder physiological conditions (10 mM PBS buffer, pH 7.4, 37 degreesCelsius) for 48 hours.

FIGS. 4 and 5 illustrate higher and extended NO release rates, which maybe achieved in part due to a higher thiol conversion. Higher andextended NO release rates enable a smaller mass of material to be usedto achieve similar results. This is important for applications wherehaving small thicknesses is important to prevent (i.e. stent coatings)or for applications where thin coatings are needed to preserve theunderlying mechanical properties of the device but thin coatings couldlimit drug loading.

Example 4 In Vitro Degradation Profiles of the S-Nitrosated PLGHPolymers

In vitro degradation profiles of the S-nitrated PLGH polymers of Example1 were determined by treating the polymers with PBS buffer at 37° C. Thedegradation profile was monitored by following the percentage weightloss. Buffer solutions were replaced weekly and polymer samples wereseparated, washed with DI water and dried under vacuum till reaching aconstant weight. FIG. 6 illustrates the In vitro degradation profile ofPLGH, PLGH-cysteine, PLGH-cysteamine, and PLGH-homocysteine polymers in10 mM PBS buffer (pH 7.4, 37° C.).

For a wide range of applications, materials are needed that providelocal drug delivery (i.e., NO release) to modulate cell/proteinbehaviors, but are then no longer needed. Thus, a material that can bothprovide local drug delivery and degradation are important. The NOrelease profiles (FIGS. 4 and 5) are significant to control many neededbiological functions. FIG. 6 shows that the material begins to degradesignificantly after the NO release is completed. For tissue engineeringor wound healing applications, the timeframe until the material beginsto degrade is ideal for supporting initial cell growth as well.

Example 5 Nitric Oxide Loading and Thiol Conversion

S-nitrosated PLGH-cysteamine, PLGH-Cysteine and PLGH-homocysteinepolymers were prepared as described in Example 1. The thiolincorporation was calculated by integrating the NMR intensities of thethiol protons and those of the PLGH backbone. The ratio of the averageNMR proton intensities between the corresponding thiol protons with thatfrom the HMPA segment of the polymer backbone gives the extent of thiolmodification to the polymers.

Nitric oxide loading was experimentally determined using UV absorptionspectroscopy. In brief, the intensity of the characteristic absorbancebetween 335 and 338 nm was measured for each polymer. The concentrationwas determined from the absorbance intensity and the molar extinctioncoefficient using Beer's Law. The extinction coefficients wereexperimentally determined for each polymer and are reported Example 1.The percent thiol conversion was calculated as the nitric oxide contentdivided by the thiol content.

Table 1 provides the thiol content and thiol conversion (i.e., nitricoxide loading).

TABLE 1 Thiol content NO loading mmol SH/g mmol SNO/g Polymer polymerpolymer % conversion PLGH-cysteamine 0.57 ± 0.03 0.53 ± 0.01 93 ± 3PLGH-cysteine 0.39 ± 0.02 0.17 ± 0.01 43 ± 1 PLGH-homocysteine 0.18 ±0.05 0.17 ± 0.01 96 ± 3High thiol conversion to form S-nitrosothiols are achieved using thenon-aqueous nitrosation reaction condition of Example 1. As shown inTable 1, PLGH-cysteamine had a thiol conversion of 93%, PLGH-cysteinehad a thiol conversion of 43%, and PLGH-homocysteine had a thiolconversion of 96%.

Example 6 Nitric Oxide Loading and Recovery

The S-nitrosated polymers of Example 5 were further tested to determinethe nitric oxide recovery.

Nitric oxide recovery was determined for each polymer using thermal drytest conditions. The samples were heated up to 100 degrees Celsius andthe nitric oxide release was determined using Sievers chemiluminescenceNO Analyzers® (NOA 280i, GE Analytical, Boulder, Colo., USA). Each testwas conducted until a baseline NO measurement was achieved.

Nitric oxide recovery for the polymer film and the polymer nanofiberunder physiological test conditions was also determined as describedabove in Example 3. Table 2 summarizes the results.

TABLE 2 NO loading NO release mmol SNO/g Thermal Polymer film NanofiberPolymer polymer mmol/g polymer mmol/g polymer mmol/g polymerS-nitrosated 0.53 ± 0.01 0.461 ± 0.016 0.241 ± 0.004 0.28 ± 0.02 PLGH-(% NO recovery: 87, (% NO recovery: 46, (% NO recovery: 52, cysteamineequivalent to 3.7 mmol equivalent to 1.96 mmol equivalent to 2.22 mmolNO/g from 4.26 mmol NO/g from 4.26 mmol NO/g from 4.26 mmol NO donor/g)NO donor/g) NO donor/g) S-nitrosated 0.17 ± 0.01 0.162 ± 0.010 (% NO0.155 ± 0.009 0.11 ± 0.01 PLGH- recovery: 97, equivalent (% NO recovery:93, (% NO recovery: 65, cysteine to 4.13 mmol NO/g equivalent to 3.96mmol equivalent to 2.77 mmol from 4.26 mmol NO NO/g from 4.26 mmol NO/gfrom 4.26 mmol donor/g) NO donor/g) NO donor/g) S-nitrosated 0.17 ± 0.010.141 ± 0.010 (% NO 0.033 ± 0.007 0.03 ± 0.01 PLGH- recovery: 83,equivalent (% NO recovery: 20, (% NO recovery: 18, homocysteine to 3.53mmol NO/g equivalent to 0.85 mmol equivalent to 0.77 mmol from 4.26 mmolNO NO/g from 4.26 mmol NO/g from 4.26 mmol donor/g) NO donor/g) NOdonor/g)

The rate of nitric oxide recovery is based on the conditions employed totrigger the release. Under thermal test conditions, nitric oxiderecovery of at least about 83% is achieved.

Example 7 Nitric Oxide Releasing Diazeniumdiolated PLGH-DETA

FIG. 7 is a scheme for preparing a NO releasing diazeniumdiolatedpolymer by reacting diethylenetriamine (DETA) with NHS activated PLGH(1) followed by treating with NO under pressure.

Example 8 NO Releasing Polymeric Nanostructures

S-nitrosated PLGH derivatives (Example 1) were electrospun in a suitablesolvent into nanofibers. These electrospun nanofibers were evaluated forNO releasing kinetics in Example 6.

DETA modified PLGH derivatives (Example 7) will be electrospun intonanofibers and will be loaded with NO under suitable pressure followedby evaluating their NO release kinetics.

Example 9 Modification of Dextran with NO Donor

Dextran modified with suitable NO donors can be prepared either byfollowing reductive amination (FIG. 8) or through using 4-nitrophenylchloroformate activation (FIG. 9). The modified dextran may be suitablefor use as biomimetic nanoscaffolds for tissue engineering.

Example 10 NO Releasing S-Nitrosated Dextran Derivatives ThroughReductive Amination

FIG. 10 is a scheme for preparing NO releasing S-nitrosated dextranderivatives through reductive amination.

S-Nitrosated Dextran-Cysteamine Through Reductive Amination (9a):

To a solution of 1 g dextran in 30 mL Millipore water, 0.8 g sodiumperiodate (3.73 mmol) was added followed by 0.3 g conc. sulfuric acid (3mmol, 0.8 eq. of periodate). The reaction mixture was protected fromdirect exposure to light and stirred for 1.5 h at room temperature.Reaction was terminated by treating with 0.18 g ethylene glycol (2.84mmol, 0.76 eq.) for half an hour and then neutralized with 0.2 M sodiumacetate solution. The resulting mass was extensively dialysed againstMillipore water using Spectra/Por® dialysis membrane (SpectrumLaboratories, CA) with a MW cut-off size of 2000 Da. Dialyzed reactionmass containing dextran aldehyde derivative (7) was cooled to 0° C. inan ice bath and 0.46 g cysteamine hydrochloride (1.05 molar eq. based onthe periodate quantity) was added. The pH of the reaction mixture wasadjusted to 8.5 by the addition of 1 M sodium hydroxide solution andstirred at 0° C. for one hour. Sodium cyanoborohydride (0.25 g, 1 eq.)was then added and the solution was stirred for further 2 h at the sametemperature. The reaction mass was neutralized with 10% acetic acid andafter an overnight dialysis with Millipore water, the dialyzed solutionwas stirred with 0.05 g dithioerythritol (DTE) for one hour at roomtemperature to reduce any disulfide formation. The solution was furtherextensively dialyzed and freeze dried to isolate the cysteamine modifieddextran derivative (8a).

S-nitrosation of the thiol terminals was performed by suspending 100 mgof the dextran-cysteamine derivative (8a) in 8 mL anhydrous methanol (80vol.) and stirred to get a uniform suspension. To this, 0.8 mLtert-butylnitrite, pre-treated with 10% w/v disodium ethylenediaminetetraacetate dehydrate (EDTA-disodium salt), was added and stirred for24 h at room temperature under an N₂ atmosphere, protected from light.Excess tert-butylnitrite was removed by washing with anhydrous methanol(3×8 mL) and dried under vacuum to isolate the S-nitrosateddextran-cysteamine derivative (9a) as an orange coloured powder.

The extent of thiol incorporation onto the dextran backbone wasquantified using Ellman's assay (Ellman, G. L. (1959). Tissue sulfhydrylgroups. Archives of Biochemistry and Biophysics, 82(1), 70-77; Frost, M.C., & Meyerhoff, M. E. (2005). Synthesis, characterization, andcontrolled nitric oxide release from S-nitrosothiol-derivatized fumedsilica polymer filler particles. Journal of Biomedical MaterialsResearch Part A, 72A(4), 409-419.) using cysteine standards. The thiolcontent was determined to be 0.16 mmol SH/g. Nitric oxide release wasdetermined as described in Example 11. The NO release was determined tobe 0.085 mmol NO/g, which is equivalent to 53% NO release, based on SH.

S-Nitrosated Dextran-Cysteine Through Reductive Amination (9b):

Experiments were performed following the method given for 9a using 0.48g cysteine (1.05 molar eq. based on the periodate quantity) andS-nitrosated following the same reaction parameters given for 8a.

The thiol content and NO release were determined as described above forS-nitrosated dextran-cysteamine through reductive amination (9a). Thethiol content was 0.263 mmol SH/g, and the NO release was 0.139 mmolNO/g (53% NO release based on SH).

Example 11 NO Release from the S-Nitrosated Dextran Derivatives ThroughReductive Amination

NO release from S-nitrosated dextran-cysteamine (9a) anddextran-cysteine (9b) of Example 10 were measurements usingchemiluminescence NO analyzer. The polymer materials were tested inphosphate buffer saline (PBS, 10 mM phosphate, pH 7.4) at 37° C. Therelease profile from S-nitrosated dextran-cysteamine (9a) anddextran-cysteine (9b) are represented in FIGS. 11 and 12 respectively.

FIGS. 11 and 12 illustrate the high NO release rates from S-nitrosateddextran-cysteamine (9a) and dextran-cysteine (9b). The NO releaseamounts are sufficient to modulate cell responses. These materials arealso degradable using enzymes.

Example 12 NO Releasing S-Nitrosated Dextran Derivatives ThroughCarboxymethylation

FIG. 13 is a scheme for preparing NO releasing S-nitrosated dextranderivatives through carboxymethylation.

Preparation of Carboxymethyl Dextran Derivative (10):

CM-dextran derivative was prepared following a modified procedurereported previously (Langmuir, 26, 7299 (2010)). Dextran (5 g) andsodium hydroxide (5 g, 125 mmol) were mixed together in 125 mL 80%2-propanol solution and stirred at 60° C. to get a clear solution. Asolution of 5.62 g chloroacetic acid (59 mmol, 0.47 eq. of NaOH) in 40mL 80% IPA solution was prepared separately and slowly charged to thereaction mass using a pressure equilibrium addition funnel over a periodof half an hour. After maintaining at 60° C. for five hour, the reactionmass was cooled to room temperature and adjusted the pH to 5 by theaddition of glacial acetic acid. The reaction mass was maintained atthis pH for one hour with occasional addition of acetic acid and slowlyquenched into methanol (1 L) and stirred for one hour to ensure thecomplete precipitation of the product. The crude product was isolated byfiltration, washed with methanol and dried under vacuum. Redissolved theproduct in 25 mL Millipore water and adjusted the pH to 5 by theaddition of glacial acetic acid (if needed) and dialyzed againstMillipore water using a MWCO membrane (2000 Da) for one week withmultiple changes per day. Finally the dialysis product was freeze-driedover 3 days to isolate the product as a white fluffy powder.

Dextran-Cysteamine Derivative (11a):

Carboxyl groups of the CM-dextran derivative (1 g, equivalent to 1.5mmol carboxyl content) was pre-activated by reacting with NHS (1.5 g,12.75 mmol, 8.5 molar eq.) and EDC.HCl (2.5 g, 12.75 mol, 8.5 molar eq.)in Millipore water (25 mL) for 30 minutes at 25° C. To this, a solutionof 3.5 g cysteamine hydrochloride (30 mmol, 20 molar eq.) was charged.Adjusted the pH to 5 by the addition of 0.1 M HCl (if needed) andstirred at 25° C. for 5 hour. Dialyzed the mass against Millipore water(pH adjusted to 5 using 0.1 M HCl) using a MWCO membrane (2000 Da) fortwo weeks with multiple changes per day and protected from directexposure to light. Finally, the product was isolated as a white fluffypowder by freeze-drying the dialyzed mass for three days.

Dextran-Cysteine Derivative (11b):

Cysteine modified dextran derivative (11b) was prepared following themethod given for 11a using 3.7 g cysteine (30 mmol, 20 molar eq.).

S-Nitrosated Dextran-Cysteamine (12a):

100 mg dextran-cysteamine (11a) was mixed with 4 mL anhydrous methanoland stirred to get a uniform suspension. 0.4 mL t-butyl nitrite wascharged and stirred the mass at 20° C. for 24 hour under N₂, protectedfrom direct exposure to light. S-nitrosated dextran derivative wasisolated by concentrating the product under vacuum, and stored under N₂at −16° C.

The thiol content was determined as described in Example 10 to be 0.21mmol SH/g.

S-Nitrosated Dextran-Cysteine (12b):

The dextran-cysteine derivative (11b) was nitrosated to yield 12bfollowing the method given for 12a.

The thiol incorporation of the dextran-cysteine derivative (11b) wascalculated by integrating the NMR intensities of the thiol protons andthose of dextran. The nitric oxide loading was determined to be 0.1412mmol NO/g polymer, which is 50% thiol conversion based on SH content,80% thiol conversion based on SNO content.

The thiol content was determined as described above in Example 10 andthe nitric oxide release was determined according to Example 13. Thethiol content was 0.281 mmol SH/g and the nitric oxide content was0.1772 mmol SNO/g, which is a 63% thiol conversion. The nitric oxiderelease was 0.141 mmol NO/g, which is a 50% NO release based on SH andan 80% NO release based on SNO.

Example 13 NO Release from the NO Releasing S-Nitrosated DextranDerivatives Through Carboxymethylation

NO release was measured using chemiluminescence NO analyzer underphysiological conditions (in phosphate buffer saline (PBS, 10 mMphosphate, pH 7.4) at 37° C.). The NO release profile from S-nitrosateddextran-cysteine (12b) of Example 12 is given in FIG. 14.

FIG. 14 demonstrates the high NO release rates from S-nitrosateddextran-cysteine (12b). FIG. 14 also illustrates sufficient NO releaseamounts to modulate cell responses in a similar manner to examples11-12. The synthesis of Example 13 provides the advantage of selectivelynitrosating only the thiol moiety for this class of polysaccharidematerials, enabling the ability to better trigger and/or extend the NOrelease capabilities when processing because the mechanisms of releaseare known. These materials are also degradable using enzymes.

Example 14 NO Releasing Chitosan Derivative

FIG. 15 is a scheme for preparing an NO releasing chitosan derivative.

Thiolated Chitosan Derivative (14):

To a solution of 0.5 g chitosan (80% deacetylated) in 100 mL 1% aceticacid solution (in Millipore water), 1.9 g EDC.HCl (10 mmol) and 1 gthioglycolic acid (10.6 mmol) was charged and stirred overnight at roomtemperature protected from light. The reaction mixture was dialyzedagainst Millipore water (pH adjusted to 5 using 0.1 M HCl) using a MWCOmembrane (2000 Da) for two days against Millipore water with multiplechanges per day. The dialyzed mass was then treated with 0.2 g DTT atroom temperature for 1 hour and then re-dialyzed for one week. Finally,the thiolated chitosan derivative (14) was isolated by freeze-drying forthree days.

S-Nitrosated Chitosan Derivative (15):

100 mg chitosan-thiol derivative (14) was mixed with 4 mL anhydrousmethanol and stirred to get a uniform suspension. 0.4 mL t-butyl nitritewas charged and stirred the mass at 20° C. for 24 hour under N₂,protected from direct exposure to light. S-nitrosated chitosanderivative was isolated by concentrating the product under vacuum, andstored under N₂ at −16° C.

Example 15 NO Release from the NO Releasing Chitosan Derivative

NO release was measured using a chemiluminescence NO analyzer underphysiological conditions. The NO release profile from S-nitrosatedchitosan derivative is given in FIG. 16. The NO release was determinedto be 0.14 mmol NO/g.

FIG. 16 illustrates that the saccharide based material of Example 15 hasextended NO release compared to its dextran counterpart (i.e., examples9-14)

Example 16 PLGH-Dextran Conjugate

Dextran modified block polymers can be prepared by chain extension byreacting PLGH (Example 1) or similar carboxyl functionalized polymerwith functionalized dextran having NO donors. One such strategy isillustrated in FIG. 17, which involves grafting dextran onto our earlyreported PLGH using the well-known carbodiimide mediated esterificationreaction. Incorporating dextran covalently onto the polymer back-boneresults in crosslinking of the polymer and provides sufficient physicalstrength to the polymer for tissue engineering applications. Inaddition, the degree of substitution (DS) on the dextran groups can beconveniently varied to optimize NO loading and release to the finalrequirement. Moreover, a favorable degradation profile is alsoanticipated by the influence of dextran hydroxyl groups on the nearbyester linkages. Because of the presence of multi-block polymericsegments with different hydrolysable characteristics, the resultingpolymer derivative will be anticipated to provide a unique andmultifaceted degradation pathway to complement their compatibility touse for various biomedical applications.

Various modifications and additions can be made to the exemplaryembodiments discussed without departing from the scope of the presentinvention. For example, while the embodiments described above refer toparticular features, the scope of this invention also includesembodiments having different combinations of features and embodimentsthat do not include all of the above described features.

The following is claimed:
 1. A polymeric composition capable ofreleasing nitric oxide and modulating biological responses, thecomposition comprising: a. a biocompatible polymer; and b. S-nitrosatedthiol bonded to the biocompatible polymer, wherein the polymericcomposition has a thiol conversion of at least 40%.
 2. The polymericcomposition of claim 1, wherein the biocompatible polymer is a syntheticpolymer selected from the group consisting of: polylactide,polyglycolide, poly(lactide-co-glycolide) and poly(ε-caprolactone). 3.The polymeric composition of claim 1, wherein the biocompatible polymeris a polysaccharide selected from the group consisting of: chitosan anddextran.
 4. The polymeric composition of claim 1, wherein theS-nitrosated thiol includes a thiol selected from the group consistingof cysteamine, cysteine, homocysteine, and combinations thereof.
 5. Thepolymeric composition of claim 1, wherein the S-nitrosated thiolincludes a thiol selected from the group consisting of glutathione andpenicillamine.
 6. The polymeric composition of claim 1, wherein thepolymeric composition has a nitric oxide loading of at least 0.1 mmol/gpolymer.
 7. The polymeric composition of claim 1, wherein the polymericcomposition consists essentially of the biocompatible polymer,S-nitrosated thiol and optionally thiol residue, and wherein thepolymeric composition has a nitric oxide recovery of at least 40% underthermal decomposition conditions.
 8. The polymeric composition of claim1, wherein the polymeric composition consists essentially of thebiocompatible polymer, S-nitrosated thiol and optionally thiol residue,and wherein the polymeric composition has a nitric oxide recovery of atleast 60% when under thermal decomposition conditions.
 9. The polymericcomposition of claim 1, wherein the polymeric composition has a nitricoxide recovery of at least 20% under physiological conditions for 48hours.
 10. The polymeric composition of claim 1, wherein thebiocompatible polymer is poly(lactide-co-glycolide), the S-nitrosatedthiol includes a thiol selected from the group consisting of cysteamine,homocysteine and combinations thereof, and the thiol conversion is atleast 90%.
 11. A polymeric composition capable of releasing nitric oxideand modulating biological responses, the composition comprising: a. abiocompatible polymer; b. thiol residue bonded to the biocompatiblepolymer, and c. S-nitrosated thiol bonded to the biocompatible polymer,wherein at least 40% of total thiol residue and S-nitrosated thiolpresent is S-nitrosated thiol.
 12. The polymeric composition of claim11, wherein the biocompatible polymer is a synthetic polymer selectedfrom the group consisting of: polylactide, polyglycolide,poly(lactide-co-glycolide) and poly(ε-caprolactone).
 13. The polymericcomposition of claim 11, wherein the S-nitrosated thiol includes a thiolselected from the group consisting of cysteamine, cysteine,homocysteine, and combinations thereof.
 14. The polymeric composition ofclaim 11, wherein the biocompatible polymer ispoly(lactide-co-glycolide) and the S-nitrosated thiol includescysteamine.
 15. The polymeric composition of claim 11, wherein thebiocompatible polymer is poly(lactide-co-glycolide) and the S-nitrosatedthiol includes cysteine.
 16. The polymeric composition of claim 11,wherein the biocompatible polymer is poly(lactide-co-glycolide) and theS-nitrosated thiol includes homocysteine.
 17. The polymeric compositionof claim 11, wherein the polymeric composition is capable of nitricoxide release over a period of at least 172 hours.
 18. The polymericcomposition of claim 11, wherein the polymeric composition has a nitricoxide recovery of at least 40% under thermal dry conditions.
 19. Thepolymeric composition of claim 11, wherein the polymeric composition hasa nitric oxide recovery of at least 20% under physiological conditionsfor 48 hours.
 20. The polymeric composition of claim 11, wherein atleast 90% of total thiol residue and S-nitrosated thiol present isS-nitrosated thiol.